Rapid development of ultrasound contrast agents in the recent years has generated a number of different formulations, which are useful in ultrasound imaging of organs and tissue of human or animal body. These agents are designed to be used primarily as intravenous or intra-arterial injectables in conjunction with the use of medical echographic equipment which employs for example, B-mode image formation (based on the spatial distribution of backscatter tissue properties) or Doppler signal processing (based on Continuous Wave or pulsed Doppler processing of ultrasonic echoes to determine blood or liquid flow parameters).
A class of injectable formulations useful as ultrasound contrast agents includes suspensions of gas bubbles having a diameter of few microns dispersed in an aqueous medium.
Use of suspensions of gas bubbles in carrier liquid, as efficient ultrasound reflectors is well known in the art. The development of microbubble suspensions as echopharmaceuticals for enhancement of ultrasound imaging followed early observations that rapid intravenous injections of aqueous solutions can cause dissolved gases to come out of solution by forming bubbles. Due to their substantial difference in acoustic impedance relative to blood, these intravascular gas bubbles were found to be excellent reflectors of ultrasound. The injection of suspensions of gas bubbles in a carrier liquid into the blood stream of a living organism strongly reinforces ultrasonic echography imaging, thus enhancing the visualisation of internal organs. Since imaging of organs and deep seated tissues can be crucial in establishing medical diagnosis, a lot of effort has been devoted to the development of stable suspensions of highly concentrated gas bubbles which at the same time would be simple to prepare and administer, would contain a minimum of inactive species and would be capable of long storage and simple distribution.
The simple dispersion of free gas bubbles in the aqueous medium is however of limited practical interest, since these bubbles are in general not stable enough to be useful as ultrasound contrast agents.
Interest has accordingly been shown in methods of stabilising gas bubbles for echography and other ultrasonic studies, for example using emulsifiers, oils, thickeners or sugars, or by entrapping or encapsulating the gas or a precursor thereof in a variety of systems. These stabilized gas bubbles are generally referred to in the art as “microvesicles”, and may be divided into two main categories.
A first category of stabilized bubbles or microvesicles is generally referred to in the art as “microbubbles” and includes aqueous suspensions in which the bubbles of gas are bounded at the gas/liquid interface by a very thin envelope involving a surfactant (i.e. an amphiphilic material) disposed at the gas to liquid interface. A second category of microvesicles is generally referred to in the art as “microballoons” or “microcapsules” and includes suspensions in which the bubbles of gas are surrounded by a solid material envelope formed of natural or synthetic polymers. Examples of microballoons and of the preparation thereof are disclosed, for instance, in European patent application EP 0458745. Another kind of ultrasound contrast agent includes suspensions of porous microparticles of polymers or other solids, which carry gas bubbles entrapped within the pores of the microparticles. The present invention is particularly concerned with contrast agents for diagnostic imaging including an aqueous suspension of gas microbubbles, i.e. microvesicles which are stabilized essentially by a layer of amphiphilic material.
Microbubbles suspensions are typically prepared by contacting powdered amphiphilic materials, e.g. freeze-dried preformed liposomes or freeze-dried or spray-dried phospholipid suspensions, with air or other gas and then with aqueous carrier, agitating to generate a microbubble suspension which must then be administered shortly after its preparation.
Examples of aqueous suspensions of gas microbubbles and preparation thereof can be found for instance in U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,413,774, U.S. Pat. Nos. 5,556,610, 5,597,549, U.S. Pat. No. 5,827,504.
WO97/29783 discloses an alternative process for preparing gas microbubbles suspensions, comprising generating a gas microbubble dispersion in an appropriate phospholipid-containing aqueous medium and thereafter subjecting the dispersion to lyophilisation to yield a dried reconstitutable product. The so prepared dried products are reconstitutable in aqueous media requiring only minimal agitation. As mentioned in said document, the size of the so generated microbubbles is consistently reproducible and in practice is independent from the amount of agitation energy applied during reconstitution, being determined by the size of the microbubbles formed in the initial microbubble dispersion. The Applicant has however observed that the amount of agitation energy applied for generating the gas microbubble dispersion in the phospholipid-containing aqueous medium may be excessively high, particularly when small diameter microbubbles are to be obtained (e.g. 23000 rpm for 10 minutes, for obtaining a dispersion of bubbles having a volume mean diameter of about 3 μm). This high agitation energy may determine local overheating in the aqueous dispersion of microbubbles, which may in turn cause degradation of the phospholipids contained in the aqueous medium. In addition, the effects of an excessively high agitation energy are in general difficult to control and may result in an uncontrollable size distribution of the final microbubbles. Furthermore, this process involves a continuous flow of gas into the aqueous medium during the generation of microbubbles, thus requiring the use of relevant amounts of gases.
WO 94/01140 discloses a further process for preparing microvesicle suspensions reconstitutable in an aqueous medium, which comprises lyophilizing aqueous emulsions containing parenterally acceptable emulsifiers, non polar liquids and lipid-soluble or water-insoluble “structure-builders”. Poloxamers and phospholipids are mentioned as parenterally acceptable emulsifiers, while mixtures of these two are employed in the working examples. Cholesterol is the preferred water-insoluble structure-builder, which is employed in the working examples. The lyophilized product is then reconstituted in water, to give aqueous suspension of gas-filled microvesicles. The gas-filled microvesicles resulting from the reconstitution step are thus defined by an envelope of different materials, including emulsifiers such as poloxamers and water-insoluble structure-builders such as cholesterol.
The process is said to result into an emulsion with particles' size lower than 4 μm, preferably lower than 2 μm, down to 0.5 μm. The Applicant has however noticed that while the reconstitution step may finally result in microvesicles having a numerical mean diameter of less than 2 μm, the corresponding size distribution of the microvesicles population is nevertheless relatively broad. In addition, the conversion step from the emulsion microparticles, obtained according to the above process, into gas microbubbles results in rather low yield.
The Applicant has now found that a much narrower distribution of microbubbles size can be obtained if a phospholipid is used as the main emulsifier of the above emulsion and if the above process is conducted in the substantial absence of the above water-insoluble structure-builders. In addition, the substantial absence of said water-insoluble structure-builders allows to substantially increase the conversion yield from emulsion microparticles into gas microbubbles. The Applicant has further observed that the above process may result in a further narrower size distribution of microbubbles and in an increased yield if the phospholipid is essentially the only emulsifier present in the emulsion.
The Applicant has also found that by applying a rather low agitation energy to an aqueous-organic emulsion during the process as above specified, it is possible to obtain microbubbles having a very small diameter and reduced size distribution.